CN104792998B - Loxl2抗原用于制备甲状腺癌预示、侵袭、转移试剂盒的应用 - Google Patents

Loxl2抗原用于制备甲状腺癌预示、侵袭、转移试剂盒的应用 Download PDF

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CN104792998B
CN104792998B CN201510102845.6A CN201510102845A CN104792998B CN 104792998 B CN104792998 B CN 104792998B CN 201510102845 A CN201510102845 A CN 201510102845A CN 104792998 B CN104792998 B CN 104792998B
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李南林
凌瑞
易军
李永平
李信
杨玉庆
陈聪
杨继鑫
赵洁
任爱雄
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Abstract

本发明提供了LOXL2抗原用于制备甲状腺癌预示、侵袭、转移检测试剂盒的应用。LOXL2的表达与甲状腺癌预示、侵袭、转移、预后有相关性，不仅可作为甲状腺癌相关试验研究中癌组织的预示、侵袭、转移标志物的应用，而且可以提高病理早期预示甲状腺癌的灵敏度。因LOXL2为分泌蛋白，还可作为非侵入性的蛋白标志物，在甲状腺癌预示、疗效评估、复发监控等方面有应用的前景。

Description

LOXL2抗原用于制备甲状腺癌预示、侵袭、转移试剂盒的应用
技术领域
本发明涉及医学生物技术领域的抗体，具体的说，是赖氨酰氧化酶样蛋白2抗原用于制备甲状腺癌预示、侵袭、转移检测用试剂盒的应用。
背景技术
甲状腺癌发病率居头颈部肿瘤之首，近年来对甲状腺癌组织的预示、侵袭、转移标志物相关试验逐渐成为研究的热点。
发明内容
针对现有技术的缺陷或不足，本发明提供了LOXL2抗原用于制备甲状腺癌预示、侵袭、转移检测试剂盒的应用。
进一步提供LOXL2抗原、LOXL2单抗和LOXL2多抗用于制备甲状腺癌预示、侵袭、转移检测试剂盒的应用。
本发明的试剂盒为ELISA试剂盒。
同时还提供了一种用于甲状腺癌预示、侵袭、转移检测试剂盒，该试剂盒包括LOXL2抗原。
进一步提供的试剂盒包括：LOXL2抗原、LOXL2单抗和LOXL2多抗。
本发明的LOXL2抗的氨基酸序列为：
NFGDQGITMGCWDMYRHDIDCQWVDITDVPPGDYLFQVVINPNFEVAESDYSNNIMKCRSRYDGHRIWMYNCHIGGSFSEETEKKFEHFSGLLNNQLSP。
申请人首次发现LOXL2的表达与甲状腺癌预示、侵袭、转移、预后有相关性，不仅可作为甲状腺癌相关试验研究中癌组织的预示、侵袭、转移标志物的应用，而且可以提高病理早期预示甲状腺癌的灵敏度。因LOXL2为分泌蛋白，还可作为非侵入性的蛋白标志物，在甲状腺癌预示、疗效评估、复发监控等方面有应用的前景。
附图说明
图1是实施例1中的癌与癌旁Ratio分布图；
图2是实施例2的ROC曲线下面积；
图3是实施例3的ROC曲线下面积；
图4LOXL2蛋白相互作用预测网络图谱；Cytoscape用于分子网络预测，945066为LOXL2，947814为TP53，946724为CA125，947803为CEA,944821为HER2，945672为BRCA1，948490为BRCA1，946234为cystatinD,947429为ER,851539为PR。
以下结合附图和实施例对本发明作进一步的详细说明。
具体实施方式
赖氨酰氧化酶样蛋白2(Lysyloxidaselikeprotein2，LOXL2)是赖氨酰氧化酶(Lysyloxidase，LOX)家族中的一种依赖铜的分泌型胺氧化酶，催化细胞外基质胶原与弹力蛋白交联，使其纤维化，在保持细胞外基质结构的完整性、细胞运动、肿瘤发生发展等生物学功能方面起重要作用。
为了证明LOXL2在甲状腺癌预示、侵袭、转移标志物的实际应用，申请人采用Real-timePCR(染料法、探针法)检测甲状腺穿刺组织中LOXL2mRNA表达量，根据cutoff值，判断病理组织良恶性，辅助病理预示；并采用夹心法ELISA检测血液(血清、血浆)中LOXL2分泌蛋白含量，用于甲状腺癌预示、疗效评估、复发监控等方面的具体试验。
在以下的实施例中，缩略语、英文和关键术语定义列表：
LOXL2 Lysyloxidase like protein 2
CP Cross point
GAPDH glyceraldehyde-3-phosphate dehydrogenase
PBS 磷酸盐缓冲液
PBST 磷酸盐吐温缓冲液
TMB 3,3',5,5'-四甲基联苯胺
实施例1：Real-timePCR检测甲状腺穿刺组织中LOXL2mRNA表达量(染料法)
设计LOXL2跨外显子的引物，扩增子139bp：
上游引物：CATGGGCTGCTGGGACATG
下游引物：GTTGTTGGAGTAATCGGATTCTGC
GAPDH上、下游引物：
上游引物:GGGTGTGAACCATGAGAAGT
下游引物：GACTGTGGTCATGAGTCCT
使用商品化的新鲜组织RNA提取试剂TRIzolReagent，Invitrigen(货号：15596026)，提取的基因组RNA，取1ugRNA逆转录为cDNA(Fermentas商品化试剂盒，货号K1622)，稀释10倍后，取2ul作为PCR模板。
上、下游引物终浓度250nM(英潍捷基上海合成)，TaqDNA聚合酶(罗氏，货号12032953001)，终浓度1U/反应。UDG(NEB)，0.5U/反应。
dNTP(TAKARA)终浓度250nM，EvaGREEN1ul/反应。
PCR仪：Roche480。
反应条件：37度，2分钟；预变性95度，5分钟；扩增95度，10秒，60度，15秒，72度，20秒，40个循环。溶解95度，1分钟，40度，1分钟，65度，1秒，95度，持续，每度采集25次信号。
22例癌与癌旁配对样本，相对定量方法，2-ΔΔCP。LOXL2表达水平在癌中明显上调。癌与癌旁Ratio分布图参见图1。
实施例2：Real-timePCR检测甲状腺穿刺组织中LOXL2mRNA表达量(探针法)
LOXL2引物、探针：
上游引物：TCGCCGCCAACGCCACAACCA
下游引物：CGCATCGCTGCTCCCCGAAGAGC
探针：FAM-ACCGCCATGACATCGACTGCCAGTG-TAMRA
使用商品化的新鲜组织RNA提取试剂TRIzolReagent，Invitrigen(货号：15596026)，提取的基因组RNA，取1ugRNA逆转录为cDNA(Fermentas商品化试剂盒，货号K1622)，稀释10倍后，取2ul作为PCR模板。
上、下游引物终浓度250nM(英潍捷基上海合成),TaqDNA聚合酶(罗氏，货号12032953001)，终浓度1U/反应。UDG(NEB)，0.5U/反应。
dNTP(TAKARA)终浓度250nM；探针终浓度250nM。
PCR仪：Roche480。
反应条件：37度，2分钟；预变性预变性95度，5分钟；扩增95度，10秒，60度，30秒，45个循环。
合成LOXL2的DNA序列为：
Gccacaaggccagcttctgcttggaggacacagaatgtgaaggagacatccagaagaattacgagtgtgccaacttcggcgatcagggcatcaccatgggctgctgggacatgtaccgccatgacatcgactgccagtgggttgacatcactgacgtgccccctggagactacctgttccaggttgttattaaccccaacttcgaggttgcagaatccgattactccaacaacatcatgaaatgcaggagccgctatgacggccaccgcatctggatgtacaactgccacataggtggttccttcagcgaagagacggaaaaaaagtttgagcacttcagcgggctctta
LOXL2全长氨基酸序列：
LOXL2的氨基酸序列是：
NFGDQGITMGCWDMYRHDIDCQWVDITDVPPGDYLFQVVINPNFEVAESDYSNNIMKCRSRYDGHRIWMYNCHIGGSFSEETEKKFEHFSGLLNNQLSP
反转录为RNA，作为标准品，106，105，104，103，102，10拷贝，与样本一起扩增，做标准曲线，从曲线上读出样本的拷贝数。
30例甲状腺癌样本，30例炎症、良性增生等样本，统计cutoff值705copy，灵敏度70.4％，特异性78.9％。
ROC曲线下面积：74.3％(图2)。
ROC曲线下的面积值在1.0和0.5之间。在AUC＞0.5的情况下，AUC越接近于1，说明诊断效果越好。AUC在0.5～0.7时有较低准确性，AUC在0.7～0.9时有一定准确性，AUC在0.9以上时有较高准确性。AUC＝0.5时，说明诊断方法完全不起作用，无诊断价值。AUC＜0.5不符合真实情况，在实际中极少出现。
实施例3：双抗夹心酶联免疫吸附(ELISA试剂盒)预示甲状腺癌
该实施例所用试剂为：
LOXL2抗原、LOXL2单抗、LOXL2多抗均购自NovusBiologicals，货号分别为H00004017-Q01、H00004017-M05、NBP1-32954。
多抗标记：用HRP标记兔抗LOXL2多抗，HRP为德国BoehringerMannheim公司产品。
辣根过氧化物酶(HRP)，市售。
TMB(3,3',5,5'-四甲基联苯胺)，市售。
PBST(磷酸盐吐温缓冲液)，市售。
PBS(磷酸盐缓冲液)，市售。
检测方法：
采用过硫酸钠法，抗体和辣根过氧化物酶(HRP)的重量比例为1：1，HRP和抗体混合后置4℃条件下过夜，然后以0.02mol/L、pH7.4的PBS于4℃透析过夜，其他采用常规标记方法。
双抗夹心酶联免疫吸附(ELISA试剂盒)：0.05mol/L、pH9.6的碳酸盐缓冲液稀释包被抗体至包被浓度，加入酶标板(Corning)100μl/孔，置4℃条件过夜，甩干。
含30％小牛血清的PBST(其中含0.05％的Tween220)0.02mol/L，pH7.4，封闭200μl/孔，37℃，1小时，甩干，以PBST洗板1次；
加入样品稀释液(含10％的Trinton2100)5μl/孔，然后加入待检测血清50μl/孔，设阴、阳对照各3孔，37℃，30分钟，以PBST冲洗5次，甩干；
将HRP标记的抗体稀释后加入酶标板(50μl/孔)，37℃，30分钟，以PBST冲洗5次，甩干；加入底物液和TMB各50μl/孔，37℃，10分钟；
加入2mol/L的H2SO4(50μl/孔)，终止反应，于450nm测吸光度(A值)。
LOXL2单抗作为捕获抗体，包被单抗3ug/孔，HRP标记多抗1：1000(抗体浓度与整个试剂浓度之比)稀释，抗原标准品50pg/ml，100pg/ml，200pg/ml。
该实施例检测甲状腺患者血清30例，正常献血员血清30例，当cutoff值取86.3920pg/ml时，检测的灵敏度75.9％，特异性96.7％。ROC曲线下面积0.947(图3)。
实施例4：ELISA甲状腺癌疗效评估
LOXL2的ELISA操作同实施例3，用药前检测血清LOXL2水平，跟踪随访10位患者，疗程结束后再行检测LOXL2水平，对比LOXL2血清含量变化与治疗效果是否有相关性。结果见表1。
表1
由上表可以看出，检测LOXL2血清水平可对甲状腺癌治疗效果进行评估。
实施例5：ELISA甲状腺癌复发监控
LOXL2的ELISA操作同实施例3，对达到治疗终点的患者10例，检测LOXL2血清水平，每半年检测一次，跟踪随访2年，结果如下表2：
表2

Claims (5)

1.LOXL2抗原用于制备甲状腺癌预示、侵袭、转移检测试剂盒的应用，所述的LOXL2抗原的氨基酸序列为：
NFGDQGITMGCWDMYRHDIDCQWVDITDVPPGDYLFQVVINPNFEVAESDYSNNIMKCRSRYDGHRIWMYNCHIGGSFSEETEKKFEHFSGLLNNQLSP。
2.LOXL2抗原、LOXL2单抗和LOXL2多抗用于制备甲状腺癌预示、侵袭、转移检测试剂盒的应用，所述的LOXL2抗原的氨基酸序列为：
NFGDQGITMGCWDMYRHDIDCQWVDITDVPPGDYLFQVVINPNFEVAESDYSNNIMKCRSRYDGHRIWMYNCHIGGSFSEETEKKFEHFSGLLNNQLSP。
3.如权利要求2所述的应用，其特征在于，所述的试剂盒为ELISA试剂盒。
4.一种用于甲状腺癌预示、侵袭、转移检测试剂盒，其特征在于，该试剂盒包括LOXL2抗原，所述的LOXL2抗原的氨基酸序列为：
NFGDQGITMGCWDMYRHDIDCQWVDITDVPPGDYLFQVVINPNFEVAESDYSNNIMKCRSRYDGHRIWMYNCHIGGSFSEETEKKFEHFSGLLNNQLSP。
5.如权利要求4所述的试剂盒，其特征在于，所述试剂盒包括：LOXL2抗原、LOXL2单抗和LOXL2多抗。
CN201510102845.6A 2015-03-09 2015-03-09 Loxl2抗原用于制备甲状腺癌预示、侵袭、转移试剂盒的应用 Expired - Fee Related CN104792998B (zh)

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